The present invention relates to chelators and their use as therapeutic agents. More particularly, the present invention relates to metal chelators and their use as chemotherapeutic agents in the treatment of cancer.
Biomolecules responsible for oxygen transport, electron transport, oxidation, reduction, and diverse other functions contain iron (xe2x80x9cFexe2x80x9d) at their active sites. Iron is also essential for the catalytic activity of numerous critical enzymes, including respiratory chain enzymes and ribonucleotide reductase. Ribonucleotide reductase catalyzes the reduction of ribonucleotides to deoxynucleotides, the rate limiting step in DNA synthesis. Given the role of iron at the cellular level, in particular DNA synthesis, modulation of iron metabolism at the cellular level may play a key role in the treatment of various pathological conditions, for example, systemic iron overload and oxidative stress.
Iron deprivation strategies may indeed be useful in the treatment of cancer, a disease characterized by uncontrolled cell division. In fact, several strategies have been explored for applying iron deprivation therapy to treat cancer. For example, antibodies against transferrin receptors, which are responsible for the cellular uptake of circulating iron, have been used in the treatment of both hematopoetic and non-hematopoetic tumors. Gallium nitrate, which binds to the transferrin receptor, has been studied for use in the treatment of lymphoma and bladder cancers [Miller, R., Cancer Chemotherapy, and Pharmacology 30 Suppl. S99 (1992); Chitambar, C., Amer. J. Clin. Oncol. 20:173 (1997)]. Bladder cancer has been studied as a potential clinical target of iron depletion therapy [Seymour, G., Urol. Res. 15:341 (1987); and Seligman, P.,Amer. J. Hematol. 41:232 (1992)].
Iron chelators have been used diagnostically and as agents in the treatment of various disorders. Iron chelators are molecules that bind tightly to metal ions, rendering them chemically inert. The chemical bond formed between the chelator and the metal involves the donation of electrons present in the molecular orbital of the chelator to the vacant metal orbital. In general, a chelator can be characterized by the identity and number of donor atoms it contains, and its binding geometry. The most stable chelator metal complexes, or chelates, are formed when the denticity of the chelator is sufficient to coordinately saturate the metal. Iron occurs as a di- or trivalent cation with a coordinate number of six. Thus, stable iron chelators are advantageously hexadentate.
Iron chelators are commonly used to treat iron overload associated with genetic disorders and transfusion-dependent anemias (see U.S. Pat. No. 5,430,058). Iron chelators have also been used as contrast agents for diagnostic imaging, including X-ray and ultrasound, radiotherapy, and heavy metal detoxification (see U.S. Pat. No. 5,446,145). However, iron chelators have been developed primarily for use as agents to treat iron overload, and their effectiveness as well as the theories for their development have been based on this application. For this application, the desirable characteristics for iron chelators include chelation of Fe(III) with high stability, ability to mobilize iron for excretion, oral availability, and low chronic toxicity.
Representative iron chelators for the treatment of iron overload include members of the following five classes: hydroxamates, amine carboxylates, catechols, hydroxpyridinones and pyridoxal isonicotinoly hydrazones. Desferrioxamine (DFO), a member of the hydroxamate class of iron chelators, is the present clinical standard for the treatment of iron overload. DFO fulfills some but not all of the above-mentioned criteria for the treatment of iron overload. In particular, DFO exhibits a high and selective affinity for Fe(III). DFO is hexadentate so its effectiveness in binding iron is only weakly dependent on its concentration [Nathan, D., N. Eng. J. Med.332(14) (1995)]. DFO is also able to mobilize iron for excretion. However, DFO suffers from serious limitations. Like most hydorxymates, DFO is acid labile, displays some chronic toxicity, and cannot be given orally. Parenteral administration is required, causing both compliance problems and further limiting the drug""s utility in third world nations where iron overload is common, and facilities and supplies for parenteral administration are lacking. Further, the high cost of DFO, which must be isolated from Streptomyces cultures, further limits this drug""s utility [Hoffbrand, A., J. Lab. Clin. Med 123:492 (1994)].
The hydroxpyridinone family of chelators has recently been developed, and is currently being used in clinical trials for the treatment of iron overload. Deferiprone, also known as LI (a 1,2 dimeth-3-hydroxypyrid-4,1 compound), is a hydroxpyridinone, which has been the target of considerable study. Deferiprone is an orally active iron chelator [Olivieri, N., N. Engl. J. Med 14:918-922 (1995)] that mobilizes iron. However, deferiprone""s affinity for Fe(III) is only moderate, and this affinity has a strong dependence on the concentration of deferiprone [Loebstein, R., Clin. Drug. Invest, 13(6):345-349 (1997)]. Deferiprone has its limitations. Generally, deferiprone has a much lower therapeutic ratio than DFO. It is considerable more toxic, and has known serious side effects including agranulocytosis.
Another class of iron chelators, pyridoxal isonicotinoyl hydrazone (PIH) and its derivatives, has also been studied. PIH derivatives and their iron complexes exhibit good intracellular mobility, but their affinity for Fe(III) is only moderate [PCT WO 960253 1, Jul. 10, 1995].
Chemotherapeutic agents which exploit iron deprivation mechanisms represent a relatively unexplored field of study. These agents are considered antimetabolites, since they interfere with DNA synthesis. Some iron(III) chelators have also been studied for use as chemotherapeutic agents. Desferrioxamine (DFO) is currently being tested in clinical trials as a combination chemotherapeutic agent for treating neuroblastoma and prostate cancer [Donfranesco, A., Acta, Haematol. 95:66 (1996); and Frantz, C., Proc. Acad. Soc. Clin. Oncol.: 416 (abstr) (1994)]. Chelators of the pyridoxal isonicotinoyl hydrazide (PIH) family have also been studied as anti-proliferative agents. Members of the PIH family are tridentate ligands having both oxygen and nitrogen donor atoms. Several members of the PIH family have been identified with an IC50 (1-7 xcexcM) lower than desferioxamine (70 xcexcM), and a potential correlation between lipophilicity and cytotoxicity [Richardson, D., Blood 86:4295 (1995)].
Most chelators selectively favor iron (III), (xe2x80x9cFe (III)xe2x80x9d), due to the stronger binding and lower toxicity of iron (III) over iron (II), (xe2x80x9cFe(II)xe2x80x9d). While all of the above chelators bind iron(III), chelators of iron(II) are relatively unexplored due in part to their relative lack of metal specificity and the potential toxicity of Fe(II). Fe(II) may reduce H2O2, resulting in the production of the highly reactive, tissue-damaging hydroxyl radical. Not all chelators yield toxic Fe(II) complexes, however, because structural features of the chelator, such as steric effects, may interfere in the mechanism of hydroxyl radical formation. Also, a chelator such as phenanthroline affords a redox potential of Fe(II) (+1.15 V/NHE) too positive to allow reduction of H2O2.
Chelators of iron(II) that are redox-active may also draw on bound iron(II), if they can reduce it to iron(II). The property of redox may, therefore, confer advantages on iron(II) chelators relative to iron(III) chelators for several reasons. First, there is a pool of intracellular iron accessible to iron(II) chelators, and, second, there are cellular stores of iron(III) that may be accessed by reduction to iron(II). Thus, there is a need for chelators of greater versatility, that may access both Fe(III) and Fe(II), for clinical use.
One substance, bipy, has been found to be an intracellular chelator of Fe(II) in the treatment of vasospasm [Horky et al., PCT WO 97/49401, Dec. 31, 1997]. It is believed that this chelator functions through weak complexation of Fe(II), which allows open coordination sites to facilitate delivery of nitric oxide to walls of blood vessels. This weak complexation of Fe(II) is not suitable for action as an iron deprivation agent. Further, bipy has demonstrated only weak cytotoxic effects on tumor cells.
Another substance with Fe(II) chelating and redox properties is tachpyr. Initially discovered and synthesized by several co-inventors of the present application, tachpyr reacts with Fe(II), and causes a state of cellular iron deprivation. Tachpyr is more effective than bipy because it is a hexadentate chelator, while bipy is bidentate. Thus, tachpyr""s effectiveness as an Fe(II) chelator is independent of its concentration. Tachpyr exhibits redox properties, and binds Fe(III) through reduction to Fe(II). Tachpyr stoichiometrically forms hydroxyl radicals from hydrogen peroxide. The reductive capture of Fe(III) is believed to occur via oxidation of tachpyr to a new imine ligand, xe2x80x9ctachpyr-nH2xe2x80x9d (n=1,2,3), with concomitant reduction of Fe(III) to complexed Fe(II), xe2x80x9cFe[tachpyr-nH2]2+xe2x80x9d.
More recently, three oxygen donor groups were added to tach. The resulting chelators bind Al(III), Ga(III) and Fe(III), but no interaction with Fe(II) is described or expected [Bollinger, J. et al., Inorg Chem 33, 1241 (1994)]. 1,3,5-Triamino-1,3,5-trideoxy-cis-inositol is a tridentate chelator similar to tach. Very few hexadentate derivatives of this substance have been prepared [Hegetschweiler, et al., Inorg Chem 31, 4027 (1992)].
One of the compounds of tachpry is cis-cis-1,3,5-triaminocyclohexane or xe2x80x9ctach.xe2x80x9d Tach, a tridentate ligand, has been known for some time. Addition of three donor groups to tach forms a hexadentate ligand. A very small number of such hexadentate derivatives were known prior to this invention. Tachimpyr and Fe[tachimpyr]2+ were prepared, and not further studied [Lions et al., J. Am. Chem. Soc. 79,1572 (1957)].
There are several potential problems with the chemistry of Fe[tachpyr-nH2]2+ that may limit its iron deprivation effects and, therefore, its cytotoxicity. Fe[tachpyr-nH2]2+ and other imine complexes may be attacked by nucleophiles, leading to release of Fe. This problem is related to the susceptibility of imino groups to hydrolysis. Thus, the free chelator tachpyr-3H2 has no cytotoxicity, and is unstable in aqueous medium. Another significant issue with Fe[tachpyr-H2]2+ is its charge of +2. A positive charge generally restricts the penetration of biological membranes. Thus, tachpyr is unlikely to mobilize intracellular iron, due to inability of Fe[tachpyr-nH2]2+ to cross cell membranes. A positive charge also facilitates reactivity with biologically available anions such as chloride or hydroxide. Thus, for reasons of charge (+2) and chemical structure (imino groups), tachpyr-nH2 has intrinsic limitations in its action as an iron deprivation agent.
While chemotherapeutic agents have been in use for over fifty years, the search for new anti-cancer drugs is ongoing. There remains a general need for alternative agents, and more particularly, iron deprivation chemotherapeutic agents with novel cytotoxic mechanisms. This is so because anti-cancer drugs, which exploit different cytopathic mechanisms, can be used in combination to achieve maximum tumor effect with minimal toxicity.
The present invention is directed to a novel family of metal chelators and their metal complexes. The metal chelators are further characterized as hexadentate, chemical compounds that bind iron relatively independent of concentration, and cross the cell membrane to chelate intracellular iron pools. The metal chelators comprise a varying number of chelating moieties attached to a linking group. The chelating moiety is a linear or cyclic hydrocarbon, which is substituted at one or more carbons with a donor atom, and the linking group is substituted at one or more positions by a donor atom. The metal chelators of the present invention are represented by the general formula below: 
wherein:
X1, X3, and X5 are N, O or S, such at the X1, X3, and X5atoms are at the 1, 3, and 5 positions of a cyclohexyl group and are in a cis, cis disposition;
B, Bxe2x80x2, and Bxe2x80x3 are aliphatic, branched aliphatic, or aryl groups, or any combination thereof, wherein the number of atoms between X and Y is about 2 to about 4;
Y, Yxe2x80x2 and Yxe2x80x3 contain N, O, or S atoms that originate from either aliphatic, branched aliphatic, aryl, or heterocyclic groups, or any combination thereof, and/or Y, Yxe2x80x2 and Yxe2x80x3 are NH2 or NHR, OH, or SH, CO2H, P(O)(OH)2, RP(O)OH, ROP(O)OH groups or any combination thereof, and R is H, aliphatic, branched aliphatic, or aryl groups, or any combination thereof that may or may not be identical in Y, Yxe2x80x2 and Yxe2x80x3;
s, sxe2x80x2, and sxe2x80x3 are 0 to about 2; and
t, txe2x80x2, and txe2x80x3 are 0 to about 2.
The present invention is also directed to pharmaceuticals including chemotherapeutic agents, comprising as active ingredients the metal chelators of the general formula described above as well as the embodiments which are described below. The antiproliferative activity exhibited by the present compounds is consistent with their utility as chemotherapeutic agents. While various modes of administration are contemplated for the metal chelators as pharmaceuticals, oral administration is the preferred route of administration. The compositions can be orally administered by incorporating them with a liquid diluant or a solid carrier. As a pharmaceutical, the composition of the present invention can be used to reduce the level of iron in cells in need of such a reduction. In one embodiment, the composition of the present invention is used to inhibit tumor cell growth. While the composition of the present invention is used in the therapeutic reduction of iron, the composition may also be used to remove other metals present in amounts in need of reduction.
The metal chelators of the present invention are distinguishable in several ways from known cytotoxic iron chelators such as DFO and PIH in both their chemical structure and physical properties. The present metal chelators are characterized by a donor atom in the linking group as well as in the attached chelating moieties. The metal chelators are hexadentate ligands with profound cytotoxicity to tumor cells. While oral administration is preferable for these compounds, it is contemplated that other forms of administration may be used. The cytotoxic mechanism for the present compounds is believed to include intracellular iron chelation. Further, the chemical properties of the present compounds may allow them to bind as well as to reduce iron, which profoundly influences their biological activity.
The metal chelators of the present invention provide significant advantages and innovations in design over tachpyr and over other chelators mentioned above in a number of ways. For example, chelator tach-C(Me)-pyr (R=Me), shown in FIG. 2, is intended to provide greater redox activity relative to tachpyr, in its ability to reductively capture Fe(III). Thus, Fe[tach-C(Me)-pyr-nH2]2+ forms with greater facility relative to Fe[tachpyr-nH2]2+. It also provides steric hindrance to prevent the hydrolysis of imino groups, leading to the loss of Fe. S,S,S-tachen-2-Bn (FIG. 3) and S,S,S-tachen-2-Me (FIG. 3 ) are chelators with primary amino groups, which are better donors to Fe(II) relative to Fe(III). Greater basicity may also lead to greater affinity for Fe(II) relative to the pyridyl group of tachpyr. Chelators with a predicted greater mobility of their Fe complexes include tachcarbox, R=Me or Et (FIG. 4). This ligand will form neutral or anionic complexes with iron, which will facilitate iron mobilization.
Another property shared by the novel metal chelators tachen-OH (FIG. 4) and tachcarbox, R=Me or Et (FIG. 4) is versatility in the binding of both oxidation states of iron. Through incorporation of both nitrogen and oxygen donor atoms, which have affinity for Fe(II) and Fe(III), respectively, tachen-OH and tachcarbox, R=Me or Et may access both biologic forms of iron. Intracellular penetrating ability of the free chelators is often related to their lipophilicity. The donor atoms of tachquin (FIG. 2), are similar to tachpyr, but the additional aromatic rings of the quinoline group increase lipophilicity. Tach-6-Mepyr (FIG. 2) is another such chelator, which has 6-methyl groups to provide greater lipophilicity relative to tachpyr.
The effectiveness of the iron complexes in producing injurious hydroxyl radicals may also be enhanced relative to tachpyr. Thus, Fe-tachcarbox complexes may have a more negative redox potential relative to Fe(tachpyr-nH2), allowing more efficient cycling of Fe(III)-Fe(II) to produce hydroxyl radicals.
The chelators (Nxe2x80x94R)3tachpyr, R=Me or Et (FIG. 4) demonstrate low toxicity in this family of compounds, in that they are not effective chelators of Fe(II), nor are they cytotoxic or antiproliferative agents, yet they share some of the same moieties in their structure.
A preferred embodiment of the metal chelators of the present invention is represented by the following chemical formula: 
wherein:
X1, X3, and X5 are N, O or S, such at the X1, X3, and X5 atoms are at the 1, 3, and 5 positions of a cyclohexyl group and are in a cis, cis disposition;
B, Bxe2x80x2, and Bxe2x80x3 are aliphatic, branched aliphatic, or aryl groups, or any combination thereof, wherein the number of atoms between X and Y is about 2 to about 4;
Y, Yxe2x80x2 and Yxe2x80x3 contain N, O, or S atoms that originate from either aliphatic, branched aliphatic, aryl, or heterocyclic groups, or any combination thereof, or NH2 or NHR, OH, or SH, CO2H, P(O)(OH)2, RP(O)OH, ROP(O)OH groups or any combination thereof, and R is H, aliphatic, branched aliphatic, or aryl groups, or any combination thereof that may or may not be identical in Y, Yxe2x80x2 and Yxe2x80x3;
t, txe2x80x2, and txe2x80x3 are 0 to about 2; and
n is between 0 and about 3.
In yet another preferred embodiment, the metal chelators are represented by the following chemical formula: 
wherein:
X1, X3, and X5 are N, O or S, such at the X1, X3, and X5 atoms are at the 1, 3, and 5 positions of a cyclohexyl group and are in a cis, cis disposition; and
s, sxe2x80x2, sxe2x80x3 are 0 to about 2; 
wherein:
R is H, aliphatic, branched aliphatic, or aryl groups, or any combination thereof that may or may not be identical in Z, Zxe2x80x2 and Zxe2x80x3;
Y is NH2 or NHRxe2x80x2, OH, or SH, CO2H, P(O)(OH)2, RP(O)OH, Rxe2x80x2OP(O)OH groups, or a combination of these is Rxe2x80x2 is H, aliphatic, branched aliphatic, or aryl groups, or any combination thereof, or Y is a group containing N, O, or S atoms that originate from either aliphatic, branched aliphatic, aryl, heterocyclic groups, or any combination thereof, in any case, Y and Rxe2x80x2 may or may not be identical in Z, Zxe2x80x2 and Zxe2x80x3;
t is 0 to about 2;
n is between 0 and about 3.
In yet another preferred embodiment, a pharmaceutical composition for treating and preventing medical conditions in mammals is disclosed, comprising as active ingredient a compound of the formula: 
wherein:
X1, X3 and X5 are N, O or S, such at the X1, X3, and X5 atoms are at the 1, 3, and 5 positions of a cyclohexyl group and are in a cis, cis disposition;
B, Bxe2x80x2, and Bxe2x80x3 are aliphatic, branched aliphatic, or aryl groups, or any combination thereof, wherein the number of atoms between X and Y is about 2 to about 4;
Y, Yxe2x80x2 and Yxe2x80x3 contain N, O, or S atoms that originate from either aliphatic, branched aliphatic, aryl, or heterocyclic groups, or a combination thereof, and/or Y, Yxe2x80x2 and Yxe2x80x3 are NH2 or NHR, OH, or SH, CO2H, P(O)(OH)2, RP(O)OH, ROP(O)OH groups or a combination thereof, and R is H, aliphatic, branched aliphatic, or aryl groups, or a combination thereof that may or may not be identical in Y, Yxe2x80x2 and Yxe2x80x3;
s, sxe2x80x2, and sxe2x80x3 are 0 to about 2; and
t, txe2x80x2, and txe2x80x3 are 0 to about 2.
The pharmaceutical composition comprises an active ingredient compound, which is selected from the group of metal chelators consisting of tach-C(Me)pyr, tach-6-Mepyr, tachquin, sss-tachem-2Bn, sss-tachen-2Me, (Nxe2x80x94R)3tachpyr, tachpyr-2H2 and tach-N-Me-Im-imine. The pharmaceutical composition can be formulated in a therapeutic dosage by itself, in combination with at least one other pharmaceutical, or chemically linked to at least one other pharmaceutical. The composition can be combined with pharmaceutically acceptable carriers, diluents, stabilizers, solubilizers, lubricants, binders and the like or excipients thereof. Further, the pharmaceutical composition comprises a mammalian metabolic conjugate of the active ingredient compound.
It is contemplated that the pharmaceutical composition can be used in the treatment and prevention of the following medical conditions including, but are not limited to, cancer, inflammatory and infectious conditions, vasoreactive and vasoocclusive conditions, coronary and peripheral athlerosclerosis, parasitic diseases, neurologic and neuromuscular conditions, and viral conditions including AIDS. Additional medical conditions further include vasospasm, Parkinson""s disease, Alzeihmer""s disease, malaria, tuberculosis, arthritis, allergic and asthmatic conditions, hepatitis, coronary and peripheral vascular ischemia-reperfusion injury of blood vessels.
In another embodiment, the pharmaceutical composition is orally formulated in combination with a liquid diluant or a solid carrier.
In yet another embodiment, the pharmaceutical composition is administered in a therapeutically effective dosage, which prevents the occurrence of, reduces the rate of growth of, or diminishes the size of tumor cells or any combination therof.
The preceding and further objects of the present invention will be appreciated by those of ordinary skill in the art from a reading of the detailed description of the invention and preferred embodiments which follow, such description being merely illustrative of the present invention.